Fine particle film and producing method of the same

ABSTRACT

To provide fine particle films including fine particles which are arranged at a high density in a highly accurate and regular manner is enabled. The fine particle film is a fine particle film including a substrate and plural number of protein fine particles which are arranged on the surface of the substrate in a plane direction parallel to the surface of the substrate, wherein each of the protein fine particles has plural number of first binding sites and one or more second binding sites respectively including a condensed amino acid, and each of the first binding sites binds to other first binding site carried by an adjacent fine particle while the second binding site binds to the substrate, wherein at least a part of the condensed amino acids constituting the second binding site are substituted.

FIELD OF THE INVENTION

[0001] The present invention relates to processes for producing a fineparticle film, and particularly, relates to techniques to arrange fineparticles having the size of around several ten nanometers in a highlyaccurate and regular manner.

BACK GROUND OF THE INVENTION

[0002] Fine particles have a great ratio of the surface area to thevolume thereof, and they generally behave in a different manner frommaterials having small ratio of the surface area to the volume. Forexample, fine particles of inorganic materials such as titanium oxide,zinc oxide and the like have eliminating action of ultraviolet ray,antibacterial action, catalytic action and the like.

[0003] Among fine particles of inorganic materials, fine particleshaving a diameter in a nanometer size (superfine particles) are expectedto exhibit a quantum effect. Accordingly, industrial utilization of thefine particles has drawn attention. In particular, in respect ofsuperfine particles having the diameter in nanometer size, there is anurgent need to develop industrial manufacturing techniques of elementsutilizing the quantum effects.

[0004] Protein fine particles having the diameter of about 10 to 20 nmhave drawn attention in regard to utilization for biosensors and thelike. Particularly, among various protein fine particles, there existfine particles capable of including inorganic materials inside. Suchprotein fine particles have both features of fine particles of theinorganic materials as described above and of fine particles of aprotein.

[0005] The fine particles described hereinabove usually have distributedin the form of a colloidal solution. However, it is disadvantageous inefficient utilization of the fine particle functions in the colloidalsolution as it is. Therefore, techniques which allow industriallyefficient utilization of the functions of fine particles have beensought in which the aforementioned colloidal solution is utilized as araw material.

[0006] Conventionally, two-dimensional crystal films comprising proteinfine particles have been utilized in crystal structure analyses of aprotein by an electron microscope. In this analysis, a two-dimensionalcrystal film comprising the protein fine particles is produced byfilling a colloidal solution of the protein fine particles in a trough,and concentrating the protein fine particles on a gas-liquid interfaceof this colloidal solution. According to this process, because thetwo-dimensional crystal film is formed on the gas-liquid interface, thetwo-dimensional crystal film is liable to be disrupted throughvibration.

[0007] Thus, as a technique which can be industrially utilized in anefficient manner, methods to arrange fine particles on a substrate havebeen believed to be most efficient. Therefore, to establish techniquesfor readily forming an ideal fine particle film on a substrate with fineparticles being regularly arranged at a high density has been desired.

[0008] As a technique for arranging protein fine particles on asubstrate developed heretofore, a transfer method developed by Yoshimuraet al. (Adv. Biophys., Vol. 34, p99-107 (1997)) is explained below withreference to FIG. 12.

[0009] First, in the step shown in FIG. 12(a), a liquid 24 with proteinfine particles 45 dispersed therein is injected into a sucrose solution23 having the concentration of 2% using a syringe 25.

[0010] Next, in the step shown in FIG. 12(b), the liquid 24 is elevatedup to the surface of the sucrose solution 23.

[0011] Next, in the step shown in FIG. 12(c), the liquid 24 reached tothe gas-liquid interface first forms an amorphous film 26 of the proteinfine particles, and the protein fine particles 45 reached afterwardscome to attach beneath the amorphous film 26.

[0012] Next, in the step shown in FIG. 12(d), a two-dimensional crystalfilm 27 of the fine protein fine particles 45 is formed beneath theamorphous film 26. Then, as is illustrated in FIG. 12(d), on a film 28including the amorphous film 26 and the two-dimensional crystal film 27of the protein fine particles 45, disposed a substrate 21 (siliconwafer, carbon grid, glass substrate or the like), thereby transferringthe film 28 to the surface of the substrate 21.

[0013] However, according to the aforementioned conventional method, itis highly possible that a breakage of the film 28 occurs in the stepshown in FIG. 12(d), and it is also highly possible that a part of theprotein fine particles of the two-dimensional crystal film 27 may fallaway upon the transfer. Accordingly, there are problems involvingdifficulties in transferring a two-dimensional crystal film having agreat area to a substrate without failure.

[0014] Therefore, according to the aforementioned method of Yoshimura etal., there is disclosed a method to accelerate the transfer of proteinfine particles onto a substrate surface by treating the substratesurface with aminopropylmethoxy silane so that the substrate surface ispositively charged at pH of around 7, in instances where the protein hasnegative charge at pH of around 7. In addition, it has been alsorevealed that protein fine particles are liable to bind with each other.

[0015] However, when the state of transfer of the protein fine particlesto the substrate in the two-dimensional crystal film which was obtainedaccording to the method described above is observed, with SEM or AFM,directions of symmetric axes of the protein fine particles are revealedto be random. Such random directionality results from the sites beingrandom where the protein fine particles contact with the substrate inthe method described above. Therefore, according to the method describedabove, protein fine particles may form a comparatively aggregatedstructure, however, it is difficult to obtain a two-dimensional crystalfilm having protein fine particles arranged at a high density in ahighly accurate and regular manner, with directions of the symmetricaxes of the protein fine particles being coordinated. In other words,directional control of the crystal axis of the two-dimensional crystalfilm is extremely difficult.

DISCLOSURE OF THE INVENTION

[0016] The present invention was achieved to solve the problemsdescribed above, and an object of the present invention is to provide afine particle film with fine particles having a diameter in a nanometersize which are arranged at a high density in a highly accurate andregular manner.

[0017] To achieve the above-described object, the present inventionconcerns a fine particle film comprising a substrate and plural numberof protein fine particles which are arranged on the surface of thesubstrate in a plane direction parallel to the surface of the substrate,wherein each of the protein fine particles has plural number of firstbinding sites and one or more second binding sites respectivelycomprising a condensed amino acid, and each of the first binding sitesbinds to other first binding site carried by an adjacent fine particlewhile the second binding site binds to the substrate, wherein at least apart of the condensed amino acids constituting the second binding siteare substituted.

[0018] In addition, the present invention concerns a process forproducing a fine particle film comprising a substrate and plural numberof protein fine particles which are arranged on the surface of thesubstrate in a plane direction parallel to the surface of the substrate,wherein each of the protein fine particles has plural number of firstbinding sites comprising a condensed amino acid, and each of the firstbinding sites binds to other first binding site carried by an adjacentfine particle, said process comprising: generating a second binding sitein each of the protein fine particles by substituting a part of thecondensed amino acids constituting each of the protein fine particleswith a basic amino acid; and making the substrate bind to the secondbinding site by bringing the protein fine particles into contact with anegatively charged substrate.

[0019] Moreover, the present invention concerns a process for producinga fine particle film comprising a substrate and plural number of proteinfine particles which are arranged on the surface of the substrate in aplane direction parallel to the surface of the substrate, wherein eachof the protein fine particles has plural number of first binding sitescomprising a condensed amino acid, and each of the first binding sitesbinds to other first binding site carried by an adjacent fine particle,said process comprising: generating a second binding site in each of theprotein fine particles by substituting a part of the condensed aminoacids constituting each of the protein fine particles with an acidicamino acid; and making the substrate bind to the second binding site bybringing the protein fine particles into contact with a positivelycharged substrate.

[0020] Further, the present invention concerns a process for producing afine particle film comprising a substrate and plural number of proteinfine particles which are arranged in a plane direction parallel to thesurface of the substrate on the surface of the substrate, wherein eachof the protein fine particles has plural number of symmetric axes; eachof the protein fine particles has plural number of first binding sitescomprising a condensed amino acid; and each of the first binding sitesbinds to other first binding site carried by an adjacent fine particle,said process comprising: selecting a specified single symmetric axisamong the plural number of symmetric axes by generating a second bindingsite in each of the protein fine particles through substituting a partof the condensed amino acids constituting each of the protein fineparticles with a basic amino acid; and making the substrate bind to thesecond binding site by bringing the protein fine particles into contactwith a negatively charged substrate.

[0021] Furthermore, the present invention concerns a process forproducing a fine particle film comprising a substrate and plural numberof protein fine particles which are arranged in a plane directionparallel to the surface of the substrate on the surface of thesubstrate, wherein each of the protein fine particles has plural numberof symmetric axes; each of the protein fine particles has plural numberof first binding sites comprising a condensed amino acid; and each ofthe first binding sites binds to other first binding site carried by anadjacent fine particle, said process comprising: selecting a specifiedsingle symmetric axis among the plural number of symmetric axes bygenerating a second binding site in each of the protein fine particlesthrough substituting a part of the condensed amino acids constitutingeach of the protein fine particles with an acidic amino acid; and makingthe substrate bind to the second binding site by bringing the proteinfine particles into contact with a positively charged substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a drawing schematically illustrating the process forproducing a two-dimensional crystal film according to embodiment 1. Inthe FIG. 1, (a) illustrates a step for treating the substrate 11 to givenegative charge; (b) illustrates a step to immerse the substrate 11 intoa liquid 16 with apoferritin fine particles 15 dispersed therein; and(c) illustrates a step to remove the substrate 11 from the liquid 16.

[0023]FIG. 2 is a drawing schematically illustrating the apoferritinfine particles 15 used in this embodiment.

[0024]FIG. 3 is a drawing for explaining the step shown in FIG. 1(b) inmore detail: (a) is a cross-sectional drawing; and (b) is a top view ofthe same.

[0025]FIG. 4 is an electron microscopic photograph of thetwo-dimensional crystal film obtained according to the embodiment 1.

[0026]FIG. 5 is a drawing illustrating the structure of the apoferritinfine particles used in the embodiment 1.

[0027]FIG. 6 is a drawing illustrating the structure of the apoferritinfine particles used in the embodiment 1.

[0028]FIG. 7 is a drawing illustrating the structure of the apoferritinfine particles used in the embodiment 1.

[0029]FIG. 8 is a drawing schematically illustrating the salt bridgeformed between two apoferritin fine particles.

[0030]FIG. 9 is a drawing for explaining the step shown in FIG. 1(b) inmore detail: (a) is a cross-sectional drawing; and (b) is a top view ofthe same.

[0031]FIG. 10 is an electron microscopic photograph of thetwo-dimensional crystal film obtained according to embodiment 2.

[0032]FIG. 11 is a drawing illustrating EP-ROM: (a) is a top view; and(b) is a cross-sectional view of the same.

[0033]FIG. 12 is a drawing illustrating a process for producing aconventional fine particle film. In the Figure, (a) illustrates a stepfor injecting a liquid 24 with protein fine particles 45 dispersedtherein into a sucrose solution 23; (b) illustrates a step in which theliquid 24 is elevated; (c) illustrates a step in which the protein fineparticles 45 attach beneath the amorphous film 26; (d) illustrates astep in which the film 28 is transferred to the surface of the substrate21.

BEST EMBODIMENT FOR CARRYING OUT THE INVENTION

[0034] The present invention is explained below in detail.

[0035] According to the two-dimensional film obtained by conventionalmethods described hereinabove, the protein fine particles are arrangedin a state whose symmetric axes have random directions. Accordingly,periodic structure is not found. It is believed that this is due to theabsence of specificity of the interaction among the protein fineparticles within the two-dimensional crystals. Therefore, the proteinfine particles aggregate to minimize the surface energy thereof, leadingto the random direction of the symmetric axes of the protein fineparticles.

[0036] Accordingly, the present inventor envisaged that atwo-dimensional crystal film having a periodic structure can be producedutilizing a symmetric property of protein fine particles by arrangingthe protein fine particles in a state whose symmetric axes have aspecified direction for the substrate. In other words, it was believedthat a two-dimensional crystal film can be obtained having protein fineparticles arranged at a high density in a highly accurate and regularmanner through specific interactions of the protein fine particles bybringing all of the protein fine particles to a single direction andsuppressing free rotation of the protein fine particles, when theprotein fine particles are arranged on a substrate.

[0037] Embodiments of the present invention explained below are providedon the basis of the above speculation. Embodiments of the presentinvention are explained with reference to the accompanying drawings. Fora convenience, constitutive elements which are common in each of theembodiments are denoted by the identical reference numeral. Further, theterm “bind” referred to herein means “fix via an attraction with eachother”, unless otherwise specified.

[0038] (Embodiment 1)

[0039] In this embodiment, a process for producing a two-dimensionalcrystal film having fine particles arranged at a high density in ahighly accurate and regular manner on the surface of a substrate isexplained.

[0040] Summary of the process for producing the two-dimensional crystalfilm according to this embodiment is first explained below withreference to FIG. 1.

[0041] First, in the step illustrated in FIG. 1(a), a substrate 11 isprovided, and the surface thereof is treated to have negative charge.

[0042] Next, in the step illustrated in FIG. 1(b), a liquid 16 isprovided having apoferritin fine particles 15 dispersed therein of whichpart being positively charged. The substrate 11 is immersed in theliquid 16. Accordingly, positive charge and negative charge areattracted with each other, and thus apoferritin fine particles 15 arebound on the surface of the substrate 11 which had been treated to havenegative charge.

[0043] Next, in the step illustrated in FIG. 1(c), the substrate 11 isremoved from the liquid 16.

[0044] A two-dimensional crystal film of the apoferritin fine particles15 formed on the surface of the substrate 11 is obtained via the stepsdescribed hereinabove.

[0045] Next, each of the steps is explained in more detail.

[0046] According to this embodiment, the process of Yokokawa et al.(Heisei 11 nendo, Shin-Energy, Sangyo Gijyutsu Sogo Kaihatshu KikouShinki Sangyo Sozo-gata Teian Koubo Jigyou Kenkyuu Seika Houkoku-syo“Shinki na Display heno Ouyou wo mezashita Chameleon-gata HassyokuShisutemu no Sousei”, March, Heisei 12) is employed to form a SAM (selfassembly monolayer) film of carboxyethyltrimethoxysilane on thesubstrate 11 in order to make the surface of the substrate 11 negativelycharge in the step illustrated in FIG. 1(a).

[0047] According to this embodiment, in the step illustrated in FIG.1(b), the substrate 11 obtained in the step illustrated in the aboveFIG. 1(a) is immersed in the liquid 16 having apoferritin fine particles15 dispersed therein. The apoferritin fine particles 15 used in thisembodiment are those obtained from ferritin which had been extractedfrom a bovine organ such as spleen, liver or the like. The apoferritinfine particle 15 is a protein fine particle having an extremely highsymmetric structure, which includes a four times symmetric axis S4, athree times symmetric axis S3 and a twice symmetric axis S2. For easyunderstandings, the apoferritin fine particle 15 is herein explainedregarding as a substantially cubic body.

[0048]FIG. 2 is a drawing schematically illustrating the apoferritinfine particle 15 used in this embodiment. When viewed the apoferritin 15as a cubic, the four times symmetric axis S4 passes through each centerof the 6 faces of the cubic; the three times symmetric axis S3 passesthrough each apex of the cubic; and the twice symmetric axis S2 passesthrough the mid point of each edge of the cubic. The symmetric axes arenow explained in more detail. The four times symmetric axis S4 is anaxis passing through the sites R4 a and R4 b. The three times symmetricaxis S3 is an axis passing through the sites R3 a and R3 b. The twicesymmetric axis S2 is an axis passing through the sites R2 a and R2 b.For a reference, in this Figure, the four times symmetric axis S4, thethree times symmetric axis S3 and the twice symmetric axis S2 arerepresentatively illustrated for each one axis.

[0049] The apoferritin fine particle 15 can form two pairs of saltbridges via a divalent cation with another apoferritin fine particle 15at each site R2 in the vicinity of the twice symmetric axis S2.Moreover, the apoferritin fine particle 15 used in this embodiment issubjected to genetically engineered modification so that each site R3 inthe vicinity of the three times symmetric axis S3 has positive charge.The aforementioned salt bridge and genetically engineered modificationin the apoferritin fine particle 15 in this embodiment is explainedbelow in detail.

[0050]FIG. 3(a) and FIG. 3(b) are a cross-sectional drawing; and a topview illustrating the step shown in FIG. 1(b) in more detail. Theapoferritin fine particles 15 bind to the surface of the substrate 11 ateither one of the sites R3 in the vicinity of the three times symmetricaxis S3 by means of the electrostatic interaction through using theliquid 16 prepared as described above and the substrate 11. Upon thebinding, the apoferritin fine particles 15 are sparsely present on thesurface of the substrate 11 in a state where the three times symmetricaxis S3 directs almost perpendicular to the surface of the substrate 11.In addition, when the binding of the apoferritin fine particles 15 tothe surface of the substrate 11 proceeds, adjacent apoferritin fineparticles 15 form salt bridges B 1 with each other at the sites R2, asshown in FIG. 3(a). Therefore, the particles bind to the surface of thesubstrate 11 while forming salt bridges B 1 with each other at the sitesR2 in a state where the three times symmetric axis S3 directs almostperpendicular to the surface of the substrate 11. That is, as shown inFIG. 3(b), all apoferritin fine particles 15 are arranged in a closestmanner on the surface of the substrate 11.

[0051] The event described above occurs successively on the surface ofthe substrate 11, and thus results in the formation of a two-dimensionalcrystal film which is apparently six times symmetry viewed from the top,but is actually three times symmetry, as shown in FIG. 3(b). Theapoferritin fine particles 15 are negatively charged in their entiretyat this time, therefore, the apoferritin fine particles 15 are not boundso as to overlay the first layer of the apoferritin fine particles 15which had been deposited. Consequently, a single layered film ofapoferritin is thereby formed.

[0052]FIG. 4 is an electron microscopic photograph of thetwo-dimensional crystal film obtained according to this embodiment. Thetwo-dimensional film schematically illustrated in FIG. 3(b) is a filmwith no clearance at all. However, because actual apoferritin fineparticles 15 are almost spherical, clearance exists in the film as shownin FIG. 4.

[0053] According to this embodiment, as shown in FIG. 4, atwo-dimensional crystal film is obtained having apoferritin fineparticles 15 arranged at a high density in a highly accurate and regularmanner.

[0054] In particular, according to this embodiment, a two-dimensionalcrystal film is easily obtained having apoferritin fine particles 15arranged at a high density in a highly accurate and regular manner byimmersing the substrate 11 into the liquid 16. In other words, in theprocess according to this embodiment, any operation required for highaccuracy is not included at all when the substrate 11 is brought intocontact with the liquid 16. Therefore, it is well suited to massproduction.

[0055] Although the substrate 11 was immersed into the liquid 16 in thestep illustrated in FIG. 1(b) according to this embodiment, as is shownin the aforementioned method of Yoshimura et al., it is also possible touse a process in which a two-dimensional crystal film is transferred onthe surface of the substrate 11. In brief, any process may be used aslong as contact between the liquid 16 and the surface of the substrate11 is allowed.

[0056] Additionally, although apoferritin fine particles 15 havingextremely high symmetry are used in this embodiment, high symmetry isnot necessarily required for the fine particles. By using fine particleshaving one or more substrate-binding sites that bind to the surface ofthe substrate 11 (second binding site) and plural number of mutualbinding sites which are capable of binding with each other (firstbinding site), a two-dimensional crystal film having the fine particlesarranged at a high density in a highly accurate and regular manner canbe obtained. This is caused by the formation of a repeated structureresulting from a regular arrangement on the surface of the substrate 11for the purpose of minimizing the surface energy, as long as there existspecific interactions of protein fine particles with each other withinthe two-dimensional crystal film, even in the instances of fineparticles with almost no symmetry. Therefore, apoferritin fine particles15 are used in this embodiment, but not limited thereto in the presentinvention.

[0057] It is more preferred that fine particles having high symmetry areused such as the apoferritin fine particles 15 used in this embodiment,of course. The reason for this preference is that a two-dimensionalcrystal film with highly accurate and regular arrangement at a highdensity can be more readily obtained, and that the resultingtwo-dimensional crystal film also has extremely high symmetry. Examplesof the fine particles having extremely high symmetry which may be usedinstead of the apoferritin fine particles 15 include an approximatelyspherical protein such as Dps protein, CCMV protein and the like, forexample. These proteins can be completely similarly used in thisembodiment instead of the apoferritin fine particles 15 by thegenetically engineered modification so that they have one or moresubstrate-binding sites which bind to the surface of the substrate 11 aswell as three or more mutual binding sites which are capable of bindingwith each other.

[0058] The mutual binding site of the fine particle may be any sitewhich allows for the binding with each other by at least either one ofthe interactions among hydrophilic interactions, hydrophobicinteractions and complementarities of the dimensional standard, but notlimited to the electrostatic interaction of the salt bridge or the like.

[0059] The apoferritin fine particle 15 used in this embodiment isexplained hereinafter in detail.

[0060] The apoferritin fine particle 15 used in this embodiment is a24-mer of a subunit having the molecular weight of approximately 20,000,and are spherical protein fine particles having the external diameter ofthe 24-mer as a whole of 12 nm. In general, apoferritin is present inthe living body as ferritin. Ferritin is a complex of an apoferritinfine particle with approximately 3,000 molecules of iron oxide (Fe₂O₃)which are included in the apoferritin fine particle. The apoferritinfine particle has characteristics to include any of several inorganicmaterial particles such as metal particles, metal oxides or the like.For example, the apoferritin fine particle can include a particletherein such as gold, iron oxide, cobalt oxide or the like. Accordingly,as the apoferritin fine particle 15 for producing the two-dimensionalcrystal film as described above, those including an inorganic materialparticle.

[0061]FIG. 5 is a drawing illustrating the structure of the apoferritinfine particle used in this embodiment. The apoferritin fine particle 15used in this embodiment has, as shown in FIG. 5, a structure including:a hollow core 12 with a diameter being approximately 6 nm which iscapable of carrying an inorganic material particle; and apoferritinprotein molecules (subunits) 14 surrounding the hollow core 12.

[0062] The apoferritin fine particle 15 has an extremely highlysymmetrical structure as described in the explanation for the aboveprocess. FIGS. 5 to 7 respectively present the figures viewed from thedirections of different symmetric axes carried by the apoferritin fineparticle 15. As shown in FIGS. 5 to 7, the apoferritin fine particle 15has a four times symmetric axis S4, a three times symmetric axis S3 anda twice symmetric axis S2.

[0063] Because the apoferritin fine particle 15 has predominantlynegative charge in its entirety, apoferritin fine particles 15 repelwith each other under a neutral condition. Thus, the apoferritin fineparticles 15 do not form an aggregate and thus diffuse within the liquid16.

[0064] However, the apoferritin fine particle 15 has a site where aglutamic acid residue appears on the surface in the vicinity of thetwice symmetric axis S2 (glutamic acid appearing site) 17, as shown inFIG. 7. Therefore, two apoferritin fine particles 15 can form a bindingvia an electrostatic interaction (salt bridge) so that they face oneanother with an ion (for example, cadmium ion or the like) sandwichedbetween the respective glutamic acid appearing sites 17. The binding viaan electrostatic interaction as described above is herein referred to asa “salt bridge”.

[0065]FIG. 8 is a drawing schematically illustrating the salt bridgeformed between two apoferritin fine particles 15. As shown in FIG. 7,the glutamic acid appearing site 17 has a pair of glutamic acid residues17 a located on the surface of the site R2 in the vicinity of the twicesymmetric axis S2. The glutamic acid residue 17 a is present in eachsubunit 14 one by one. Upon forming the salt bridge, as shown in FIG. 8,a divalent cation (indicated as cadmium in the Figure) is sandwichedbetween each one of a pair of glutamic acid residues 17 a and each oneof the opposing pair of glutamic acid residues 17 a. Therefore, when asalt bridge is formed between two apoferritin fine particles 15, freerotation of the two apoferritin fine particles are respectivelyinhibited. In addition, as shown in FIG. 3(a), mutual positionalrelationship of two apoferritin fine particles 15 is fixed directing tothe same direction.

[0066] For the purpose of forming the salt bridge, it is necessary tohave a cation (for example, cadmium ion and the like) which is divalentor more multivalent added in the liquid 16. The cation is preferablyadded in the range of 5 to 10 mM. In general, divalent cation is used.

[0067] Next, genetically engineered modification subjected to theapoferritin fine particle 15 is explained. The base sequence of the DNAcoding for the apoferritin fine particle 15 is set out in SEQ ID NO: 1,whilst the amino acid sequence of the apoferritin fine particle 15 isset out in SEQ ID NO: 2. Because the apoferritin fine particle 15 isconstituted from the identical 24 subunits 14, “base sequence of the DNAcoding for the apoferritin fine particle 15” and “amino acid sequence ofthe apoferritin fine particle 15” herein mean the base sequence of theDNA coding for the subunit 14 and the amino acid sequence of the subunit14, respectively.

[0068] The amino acid sequence of native apoferritin fine particlederived from bovine liver and the base sequence of the DNA encoding thesame are known, and its tertiary structure has been also elucidated. Inparticular, native apoferritin fine particle has a channel connecting tothe inside hollow core along the three times symmetric axis S3. Thereexist amino acids having negative charge under a neutral condition(acidic amino acid) around the channel.

[0069] The subunit of the native apoferritin fine particle isconstituted from 175 amino acid residues. Among the amino acid residues,many of the acidic amino acid residues are located at the site R3 in thevicinity of the three times symmetric axis S3. The first to eighth aminoacid residues of the native apoferritin fine particle are deleted uponprocessing in vivo. Therefore, the base at position 25 in the basesequence of the DNA of the apoferritin fine particle 15 of thisembodiment (SEQ ID NO: 1) is denoted as a base at the first position,whilst tyrosine at position 9 is denoted as an amino acid at the firstposition in the amino acid sequence thereof (SEQ ID NO: 2).

[0070] The apoferritin fine particle 15 used in this embodiment isproduced by using any known genetic recombination technique and proteinexpression method on the basis of the base sequence of the DNA set outin SEQ ID NO: 1. The apoferritin fine particle 15 includes amino acidsubstitution at amino acids located in the site R3 in the vicinity ofthe three times symmetric axis S3 with an amino acid having positivecharge under a neutral condition (basic amino acid). Specifically, asset out in SEQ ID NO: 1 and 2, lysine at positions 112 and 113 in theamino acid sequence of the apoferritin fine particle 15 is respectivelysubstituted from alanine and glutamine. Thus, the surface of the site R3in the vicinity of the three times symmetric axis S3 has stronglypositive charge. Accordingly, the apoferritin fine particle 15 binds tothe surface of the substrate 11 which is negatively charged at the siteR3 in the vicinity of the three times symmetric axis S3 via anelectrostatic interaction.

[0071] Although the amino acids at positions 112 and 113 herein aresubstituted with lysine, other basic amino acids are also permitted aslong as they are basic (i.e., arginine and histidine). However, in lightof the electrostatic interaction with the substrate which is negativelycharged, substitution with lysine at any of the positions is desiredamong these basic amino acids. In addition, the amino acid at position112 and the amino acid at position 113 may be the same or different.Moreover, although substitution of the amino acids at positions 112 and113 with a basic amino acid such as lysine was conducted in conjunctionwith the use of negatively charged substrate, to substitute the aminoacids at positions 112 and 113 with an acidic amino acid (i.e., glutamicacid, aspartic acid, glutamine, asparagine, particularly glutamic acid,aspartic acid among these) having the counter polarity is allowedaccompanied by using a positively charged substrate. Also in thisinstance, the amino acid at position 112 and the amino acid at position113 may be the same or different. This aspect is similarly applied tothe following embodiment 2 and below.

[0072] Meanwhile, positive charge and negative charge are presentadmixed on the surface of other sites than the site R3 in the vicinityof the three times symmetric axis S3 of the apoferritin fine particle15, with negative charge being predominant in its entirety. Thus, in thestep illustrated in the above FIG. 1(b), repulsive force acts betweenthe surface of other sites than the site R3 in the vicinity of the threetimes symmetric axis S3 of the apoferritin fine particle 15 and thesurface of the negatively charged substrate 11. Therefore, the threetimes symmetric axis S3 of the apoferritin fine particle 15 is fixedalmost perpendicularly to the surface of the substrate 11.

[0073] In the step illustrated in FIG. 1(b), the pH of the liquid 16 ispreferably adjusted to be in the range of 3 or greater and 11 or less sothat the surface of the substrate 11 has negative charge and that thesite R3 of the apoferritin fine particle 15 also has negative charge inthe liquid 16. It is particularly preferred that the pH of the liquid 16is adjusted to be in the range of 5 or greater and 10 or less.

[0074] Furthermore, in the step illustrated in the above FIG. 1(b), itis preferred that a liquid 16 including sufficiently diluted apoferritinfine particle 15 is prepared so that many cores are not generated whichcan originate the formation of a two-dimensional crystal film.Practically, it is preferred that the concentration of the apoferritinfine particle 15 in the liquid 16 is 100 μg/ml or less, and morepreferably 30 μg/ml or less.

[0075] Alternatively, instead of using the liquid 16, a method in whicha dispersion medium containing no apoferritin fine particle 15 isprovided, and thereto immersed the substrate 11, followed by adding acolloidal solution containing the apoferritin fine particles 15 at ahigh concentration to this dispersion medium in an extremely gentlemanner (for example, giving the final concentration of the apoferritinfine particle of 100 μg/ml over 10 hours) may be also employed.

[0076] Although the two-dimensional crystal film was formed on thesurface of the substrate 11 in this embodiment, but not limited thereto.For example, it is possible to form the two-dimensional crystal filmalso on the surface of a liquid, liquid crystal or the like.

[0077] Furthermore, the surface of the substrate 11 is treated so thatthe apoferritin fine particles 15 are bound via an electrostaticinteraction according to this embodiment. However, it may be constitutedto utilize any other interaction (for example, hydrophilic interaction,hydrophobic interaction and complementarity of the dimensionalstandard), which is not limited to the electrostatic interaction.

[0078] Moreover, in the instance of using other protein fine particleinstead of the apoferritin fine particle 15, the combination of theamino acid at the site to be bound to the surface of the substrate 11and the charge of the surface of the substrate 11 is preferably selectedto provide a combination of a basic amino acid and negative charge, orof an acidic amino acid and positive charge. These combinations resultin facilitated binding between the protein fine particle and the surfaceof the substrate 11 via an electrostatic interaction under the pHcondition described above.

[0079] When an amino acid which has repulsing charge to the surfacecharge of the substrate 11 is included at the site where the proteinfine particles used are bound to the substrate 11, it is preferred thatprotein fine particles including genetically engineered substitution ofa basic amino acid with an acidic amino acid are used instead so thatbinding is achieved via an electrostatic interaction.

[0080] In accordance with this embodiment, the substrate 11 with a SAMfilm formed on its surface is used. The SAM film is a single molecularfilm formed on the surface of the substrate 11, and can control thedistance to the apoferritin fine particles 15 in a highly accuratemanner. The surface of the substrate 11 may be covered with a singlemolecular film other than the SAM film. Further, a lipid bilayer film(LB film) comprising a phospholipid may be formed on the substrate 11.

[0081] (Embodiment 2)

[0082] In this embodiment, a process for producing a two-dimensionalcrystal film with fine particles being arranged at a high density in ahighly accurate and regular manner on the surface of the substrate bybringing the site in the vicinity of the four times symmetric axis S4 ofthe apoferritin fine particle 15 bound to the surface of the substrate11 is explained. This embodiment has the almost same constitution asthat of the embodiment 1 as described above, and the summary of theprocess for producing the two-dimensional crystal film according to thisembodiment is as illustrated in FIG. 1. However, the amino acid sequenceof the apoferritin fine particle 15 is different.

[0083] The apoferritin fine particle 15 of this embodiment can form twopairs of salt bridges via a divalent cation with another apoferritinfine particle at each site R2 in the vicinity of the twice symmetricaxis S2. Moreover, the apoferritin fine particle 15 used in thisembodiment is subjected to genetically engineered modification so thateach site R4 in the vicinity of the four times symmetric axis S4 haspositive charge under a neutral condition.

[0084] Specifically, the apoferritin fine particle 15 of this embodimentis produced by using any known genetic recombination technique andprotein expression method on the basis of the base sequence of the DNAset out in SEQ ID NO: 3. The apoferritin fine particle 15 includes theamino acid substitution at amino acids located in the site R4 in thevicinity of the four times symmetric axis S4 with an amino acid havingpositive charge under a neutral condition (basic amino acid). Inparticular, as set out in SEQ ID NO: 3 and 4, lysine at positions 149and 151 in the amino acid sequence of the apoferritin fine particle 15is respectively substituted from alanine and glutamine. Thus, thesurface of the site R4 in the vicinity of the four times symmetric axisS4 has strongly positive charge. Accordingly, the apoferritin fineparticle 15 binds to the surface of the substrate 11 which is negativelycharged at the site R4 in the vicinity of the four times symmetric axisS4.

[0085] Meanwhile, positive charge and negative charge are presentadmixed on the surface of other sites than the site R4 in the vicinityof the four times symmetric axis S4 of the apoferritin fine particle 15,with negative charge being predominant in its entirety. Thus, repulsiveforce acts between the surface of other sites than the site R4 in thevicinity of the four times symmetric axis S4 of the apoferritin fineparticle 15 and the surface of the negatively charged substrate 11.Therefore, the four times symmetric axis S4 of the apoferritin fineparticle 15 is fixed almost perpendicularly to the surface of thesubstrate 11.

[0086]FIG. 9(a) and FIG. 9(b) are a cross-sectional drawing and a topview for explaining the step shown in FIG. 1(b) in more detail. In thestep illustrated in FIG. 1 (b), binding via an electrostatic interactionis executed with the substrate 11 at either one of the sites R4 in thevicinity of the four times symmetric axes S4 through using the liquid 16prepared as described above and the substrate 11. Upon the binding, theapoferritin fine particles 15 are sparsely present on the surface of thesubstrate 11 in a state where the four times symmetric axis S4 directsalmost perpendicular to the surface of the substrate 11 as illustratedin FIG. 9(a). In addition, when the binding of the apoferritin fineparticles 15 proceeds, adjacent apoferritin fine particles 15 form saltbridges B1 with each other at the site R2, as shown in FIG. 9(b).Therefore, the particles bind to the surface of the substrate 11 whileforming salt bridges B1 with each other at the sites R2 in a state wherethe four times symmetric axis S4 directs almost perpendicular to thesurface of the substrate 11. This event occurs successively on thesurface of the substrate 11, and thus results in the formation of atwo-dimensional crystal film which is four times symmetry, as shown inFIG. 9(b).

[0087]FIG. 10 is an electron microscopic photograph of thetwo-dimensional crystal film obtained according to this embodiment. Thetwo-dimensional crystal film schematically illustrated in FIG. 9(b) is afilm including apoferritin fine particles 15 arranged in a checkeredpattern. However, actual apoferritin fine particle 15 is almostspherical. Therefore, spaces among each of the apoferritin fineparticles 15 are diminished as shown in FIG. 10.

[0088] Although processes for producing two-dimensional crystal filmswhich are three times symmetric and four times symmetric hereinabove,the present invention is not limited thereto. When the site R2 in thevicinity of the twice symmetric axis S2 is bound to the surface of thesubstrate 11, and apoferritin fine particles having three or more mutualbinding sites are provided, it is also possible to produce a twicesymmetric two-dimensional crystal film by the process illustrated inFIG. 1. Therefore, when a protein such as apoferritin having pluralnumber of symmetric axes is used, directions of the protein to thesubstrate can be changed. More specifically, when the direction of theprotein to the substrate is intended to direct as illustrated in FIG.3(b), substitution with a basic amino acid at the sites R3 shown in FIG.2 may be conducted. When the direction of the protein to the substrateis intended to direct as illustrated in FIG. 9(b), substitution with abasic amino acid at the sites R4 shown in FIG. 2 may be conducted. Withregard to the twice symmetric axis not shown in the Figure, substitutionwith a basic amino acid at the sites R2 shown in FIG. 2 may beconducted. The directions of a protein to the substrate can be changedeven though the same protein is used, when a negatively chargedsubstrate (positively charged substrate in the instances where sites R2to R4 are acidic amino acids) is thereafter brought into contact. It isalso enabled to alter the function of a fine particle film accordingly.

[0089] (Embodiment 3)

[0090] According to this embodiment, a method of utilizing thetwo-dimensional crystal film produced in the embodiments 1 and 2 asdescribed above is explained.

[0091] First, examples of the utilization of the two-dimensional crystalfilm produced in the embodiments 1 and 2 as described above includefloating-gates such as EP-ROM, E2P-ROM and the like. FIGS. 11(a) and (b)are a top view and a cross-sectional view of EP-ROM in which thetwo-dimensional crystal film produced according to the processdemonstrated in the embodiment 1 described above is used as afloating-gate.

[0092] In the two-dimensional crystal film produced in the embodiment 1as described above, metal particles 12 a which are included in theapoferritin fine particles 15 are arranged at high density in a highlyaccurate and regular manner with an insulated state each other. Thus, toprecisely control number of the metal particles arranged on aninsulation film is enabled by patterning and the like of atwo-dimensional crystal film 100. The electrical charge accumulated onthe floating-gate can be preciously controlled by precisely controllingthe number of metal particles. Therefore, scattering of the variationamount Vth of threshold voltage for each memory cell before and afterthe writing can be suppressed. When scattering of the variation amountVth of threshold voltage for each memory cell before and after thewriting is suppressed, the variation amount Vth of threshold voltage canbe set to be smaller than ever before. In other words, EP-ROM andE2P-ROM can be obtained which are capable of operating at lower voltagethan ever before.

[0093] Although the two-dimensional crystal film 100 may be patterned asdescribed above in order to preciously control the number of the metalparticles arranged on the insulation film, other methods may be alsoemployed. In particular, a silicon substrate covered with an insulationfilm may be used as the substrate 11 in the embodiment 1 as describedabove, and among the surface of the insulation film, an area to bepositively charged may be a regular hexagon. Thus, the apoferritin fineparticles 15 are thereby arranged at a high density in a highly accurateand regular manner along the regular hexagon. Therefore, the number ofthe metal particles 12 a included in the apoferritin fine particles 15can be preciously controlled depending on the size of area of theregular hexagon.

[0094] Further, the two-dimensional crystal films produced in theembodiments 1 and 2 as described above can be also utilized forproducing a magnetic disc.

[0095] In particular, a two-dimensional crystal film is produced usingapoferritin fine particles 15 including a magnetic material particletherein, in the embodiments 1 and 2 as described above. Next, theapoferritin fine particles 15 are removed from thus resultingtwo-dimensional crystal film by e.g., heat treatment or the like toleave the inside magnetic material alone. Accordingly, a magnetic discwith high memory density is obtained which includes magnetic materialparticles arranged at a high density in a highly accurate and regularmanner on the surface of the recording surface.

[0096] Furthermore, the two-dimensional crystal film produced in theembodiments 1 and 2 as described above can be also utilized in producinga biosensor such as a nucleotide sensor.

[0097] In particular, a two-dimensional crystal film is produced usingapoferritin fine particles 15 including a gold particle in theembodiments 1 and 2 as described above. Next, the apoferritin fineparticles 15 are removed from thus resulting two-dimensional crystalfilm to leave the inside gold particles alone. By binding a thiol DNA tothese gold particles, a nucleotide sensor with extremely high SN ratiocan be obtained having the thiol DNA arranged at a high density in ahighly accurate manner.

[0098] According to the present invention, a two-dimensional crystalfilm with fine particles having a diameter of nanometer size which arearranged at a high density in a highly accurate and regular manner isobtained.

INDUSTRIAL APPLICAPILITY

[0099] As described hereinabove, the two-dimensional crystal filmcomprising protein fine particles according to the present invention isuseful in crystal structure analyses of a protein by an electronmicroscope. Furthermore, the two-dimensional crystal film according tothe present invention is also useful in producing floating-gates such asEP-ROM and E2P-ROM, and magnetic discs.

FREE TEXT OF SEQUENCE LISTING

[0100] <223> of SEQ ID NO: 1: recombinant DNA of liver apoferritin ofEquus cebellus

[0101] <223> of SEQ ID NO: 2: recombinant liver apoferritin of Equuscebellus

[0102] <223> of SEQ ID NO: 3: recombinant DNA of liver apoferritin ofEquus cebellus

[0103] <223> of SEQ ID NO: 4: recombinant liver apoferritin of Equuscebellus

What is claimed is:
 1. A fine particle film comprising a substrate andplural number of protein fine particles which are arranged on thesurface of said substrate in a plane direction parallel to the surfaceof said substrate, wherein each of said protein fine particles hasplural number of first binding sites and one or more second bindingsites respectively comprising a condensed amino acid, each of said firstbinding sites binds to other first binding site carried by an adjacentfine particle, said second binding site binds to said substrate, and atleast a part of the condensed amino acids constituting said secondbinding site are substituted.
 2. The fine particle film according toclaim 1 wherein at least a part of the condensed amino acidsconstituting said second binding site is a basic amino acid.
 3. The fineparticle film according to claim 2 wherein said substrate is negativelycharged.
 4. The fine particle film according to claim 1 wherein at leasta part of the condensed amino acids constituting said second bindingsite is an acidic amino acid.
 5. The fine particle film according toclaim 4 wherein said substrate is positively charged.
 6. The fineparticle film according to claim 1 wherein said plural number of proteinfine particles are arranged regularly on the surface of said substrate.7. The fine particle film according to claim 1 wherein each of saidprotein fine particles has a symmetric axis.
 8. The fine particle filmaccording to claim 7 wherein said symmetric axis is a four timessymmetric axis.
 9. The fine particle film according to claim 7 whereinsaid symmetric axis is a three times symmetric axis.
 10. The fineparticle film according to claim 7 wherein said symmetric axis is atwice symmetric axis.
 11. The fine particle film according to claim 1wherein each of the first binding sites carried by adjacent two fineparticles binds via an ionic bond.
 12. The fine particle film accordingto claim 10 wherein each of the first binding sites carried by adjacenttwo fine particles binds via an ionic bond with a cation which isdivalent or more multivalent being sandwiched therebetween.
 13. The fineparticle film according to claim 1 wherein said protein is apoferritinor CCMV protein.
 14. A process for producing a fine particle filmcomprising a substrate and plural number of protein fine particles whichare arranged on the surface of said substrate in a plane directionparallel to the surface of said substrate, wherein each of said proteinfine particles has plural number of first binding sites comprising acondensed amino acid, and each of the first binding sites binds to otherfirst binding site carried by an adjacent fine particle, said processcomprising: generating a second binding site in each of said proteinfine particles by substituting a part of the condensed amino acidsconstituting each of said protein fine particles with a basic aminoacid; and making said substrate bind to said second binding site bybringing said protein fine particles into contact with a negativelycharged substrate.
 15. A process for producing a fine particle filmcomprising a substrate and plural number of protein fine particles whichare arranged on the surface of said substrate in a plane directionparallel to the surface of said substrate, wherein each of said proteinfine particles has plural number of first binding sites comprising acondensed amino acid, and each of the first binding sites binds to otherfirst binding site carried by an adjacent fine particle, said processcomprising: generating a second binding site in each of said proteinfine particles by substituting a part of the condensed amino acidsconstituting each of said protein fine particles with an acidic aminoacid; and making said substrate bind to said second binding site bybringing said protein fine particles into contact with a positivelycharged substrate.
 16. A process for producing a fine particle filmcomprising a substrate and plural number of protein fine particles whichare arranged on the surface of said substrate in a plane directionparallel to the surface of said substrate, wherein each of said proteinfine particles has plural number of symmetric axes, and each of saidprotein fine particles has plural number of first binding sitescomprising a condensed amino acid, and each of the first binding sitesbinds to other first binding site carried by an adjacent fine particle,said process comprising: selecting a specified single symmetric axisamong said plural number of symmetric axes by generating a secondbinding site in each of said protein fine particles through substitutinga part of the condensed amino acids constituting each of said proteinfine particles with a basic amino acid; and making said substrate bindto said second binding site by bringing said protein fine particles intocontact with a negatively charged substrate.
 17. The process forproducing a fine particle film according to claim 16 wherein said secondbinding site is located on said specified single symmetric axis.
 18. Theprocess for producing a fine particle film according to claim 16 whereinsaid protein is apoferritin or CCMV protein.
 19. A process for producinga fine particle film comprising a substrate and plural number of proteinfine particles which are arranged on the surface of said substrate in aplane direction parallel to the surface of said substrate, wherein eachof said protein fine particles has plural number of symmetric axes, andeach of said protein fine particles has plural number of first bindingsites comprising a condensed amino acid, and each of the first bindingsites binds to other first binding site carried by an adjacent fineparticle, said process comprising: selecting a specified singlesymmetric axis among said plural number of symmetric axes by generatinga second binding site in each of said protein fine particles throughsubstituting a part of the condensed amino acids constituting each ofsaid protein fine particles with an acidic amino acid; and making saidsubstrate bind to said second binding site by bringing said protein fineparticles into contact with a positively charged substrate.
 20. Theprocess for producing a fine particle film according to claim 19 whereinsaid second binding site is located on said specified single symmetricaxis.
 21. The process for producing a fine particle film according toclaim 19 wherein said protein is apoferritin or CCMV protein.